1. Field of the Invention
The present invention relates to DNA sequences coding for proteins useful in cell synthesis of polyhydroxyalkanoic acids (PHA). These DNA sequences can be expressed in transformed micro-organisms and plants to product polyhydroxyalkanoates (PHAs).
2. Description of Related Art
The production of intracellular polyesters belonging to the class of polymers known as polyhydroxyalkanoates (PHAs) has been observed in a wide array of prokaryotic organisms (Anderson and Dawes (1990) Microbiol. Rev. 54:450). The monomers composing the polyesters range in length from C4 (3-hydroxybutyrate) to C12 (3-hydroxydodecanoate) (Lageveen et al. (1988) Appl. Env. Microbiol. 54:2924). This class of polyesters has attracted much attention as a potential alternative of conventional petrochemical-derived plastics.
PHAs are broadly characterized according to the monomers that constitute their backbone. Polymers composed of C4-C5 units are classified as short chain lengths (scl) PHAs; polymers containing monomers of C6 units and above are classified as medium chain length (mcl) PHAs. The primary structure of the polymer influences the physical properties of the polyester.
The metabolic pathways leading to the formation of PHAs have not been elucidated for all organisms. The most extensively studied PHA biosynthetic pathway is that of Alcaligenes eutrophus (Peoples et al. (1989) J. Biol. Chem. 264:15298 and Valentin et al. (1995) Eur. J. Biochem. 227:43). This organism is capable of forming either a homopolymer of C4 (polyhydroxybutyrate, PHB) or a co-polymer of C4-C5 (PHB-PHV, polyhydroxybutyrate-polyhydroxyvalerate) (Koyama and Doi (1995) Biotechnol. Lett. 17:281). Hence, A. eutrophus is classified as a scl PHA organism. Similarly, Pseudomonas species generate a polymer composed of monomers ranging in length from C6 to C12 (Timm and Steinbxc3xcchel (1990) Appl. Environ. Microbiol. 56:3360 and Lageveen et al. (1988) Appl. Environ. Microbiol. 54:2924), and are classified as mcl PHA organisms.
The polymerization of the hydroxyacyl-CoA substrates is carried out by PHA synthases. The substrate specificity of this class of enzyme varies across the spectrum of PHA producing organisms. This variation in substrate specificity of PHA synthases is supported by indirect evidence observed in heterologous expression studies (Lee et al. (1995) Appl. Microbiol. Biotechnol. 42:901 and Timm et al. (1990) Appl. Microbiol. Biotech. 33:296). Hence, the structure of the backbone of the polymer is strongly influenced by the PHA synthase responsible for its formation.
Fluorescent pseudomonads belonging to the rRNA homology group I can synthesize and accumulate large amounts of polyhydroxyalkanoic acids (PHA) composed of various saturated and unsaturated hydroxy fatty acids with carbon chain lengths ranging from 6 to 14 carbon atoms (Steinbxc3xcchel and Valentin (1992) FEMS Microbiol. Rev. 103:217). PHA isolated from these bacteria also contains constituents with functional groups such as branched, halogenated, aromatic or nitrile side-chains (Steinbxc3xcchel and Valentin (1995) FEMS Microbiol Lett. 128:219). The composition of PHA depends on the PHA polymerase system, the carbon source, and the metabolic routes (Anderson and Dawes (1990) Microbiol. Rev. 54:450; Eggink et al. (1992) FEMS Microbiol. Rev. 105:759; Huisman et al. (1989) Appl. Microbiol Biotechnol. 55:1949; Lenz et al. (1992) J. Bacteriol. 176:4385; Steinbxc3xcchel and Valentin (1995) FEMS Microbiol. Lett. 128:219). In P. putida, at least three different metabolic routes occur for the synthesis of 3-hydroxyacyl coenzyme A thioesters, which are the substrates of the PHA synthase (Huijberts et al. (1994) J. Bacteriol. 176:1661); (i) xcex2-oxidation is the main pathway when fatty acids are used as carbon source; (ii) De novo fatty acid biosynthesis is the main route during growth on carbon sources which are metabolized to acetyl-CoA, like gluconate, acetate or ethanol; and (iii) Chain elongation reaction, in which acyl-CoA is condensed with acetyl-CoA to the two carbon chain extended 13-keto product which is then reduced to 3-hydroxyacyl-CoA. This latter pathway is involved in PHA-synthesis during growth on hexanoate.
Due to the extended homologies of the primary structures of PHAMCL synthases to the PHASCL synthases (Steinbxc3xcchel et al. (1992) FEMS Microbiol Rev. 103:217), which occur in bacteria accumulating polyhydroxybutyric acid such as e.g., Alcaligenes eutrophus, it seems likely that the substrate of PHAMCL synthases is (R)-3-hydroxyacyl-CoA. The main constituent of the polyester of P. putida KT2442 from unrelated substrates such as gluconate is 3-hydroxydecanoate; whereas 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, 3-hydroxydodecenoate, 3-hydroxytetradecanoate, and 3-hydroxytetradecenoate occur as minor constituents (Huijberts et al. (1994) J. Bacteriol. 176:1661). Thus, to serve as substrates for the PHA polymerase, the hydroxyacyl residues of the acyl-carrier proteins are most probably converted to the corresponding CoA-derivatives. This can be mediated in a one-step reaction by an (R)-3-hydroxyacyl (ACP to CoA) transferase. Alternatively, the acyl group transfer could be at a different functional group level such as 3-keto or the straight chain. The resulting acyl-CoA would then be converted to the 3-hydroxy level by the action of other enzymes as depicted in FIG. 1. Another possibility, in addition to direct transfer, would be a 2-step process with the release of the free fatty acid by a thioesterase and the subsequent activation to the CoA derivative. This could occur at any level of acyl-ACP as illustrated in FIG. 1.
Elucidation of the protein(s) involved in this conversion is of practical importance because such enzymes are potentially useful in metabolic engineering of recombinant organisms to produce PHAs. For example, expression of (R)-3-hydroxyacyl transferase in the seed of an oil-producing plant (e.g. canola or soybean) would allow transfer of acyl groups directly from lipid synthesis (in which they are ACP-linked) to polymer production (in which they are CoA-linked). Alternatively, a thioesterase and ligase, used consecutively, could accomplish the same reaction.
The isolation of phaG described herein is an example of the isolation of an enzyme that links lipid synthesis to polymer production. The methods employed provide a model for the isolation of such genes in bacteria.
The present invention provides an isolated DNA fragment comprising a nucleotide sequence encoding a protein that is involved in the linkage between fatty acid biosynthesis and PHA production. This fragment comprises a phaG gene from Pseudomonas putida KT2440 that encodes a protein shown to be critical in the production of PHA in Pseudomonas putida KT2440 when this organisms is grown on a simple carboyhydrate substrate (e.g. gluconate). This gene, termed xe2x80x9cphaGxe2x80x9d, and the PhaG protein encoded thereby, as well as biologically functional equivalents thereof, respectively, can be used in conjunction with other PHA biosynthetic enzymes in the production of novel co-polymers of PHA in both prokaryotic and eukaryotic organisms, including plants. Transformed bacteria and transgenic plants comprising and expressing this gene or its equivalents along with other PHA biosynthetic genes such as, but not limited to, a gene encoding a PHA synthase, will be able to form hydroxyacyl-CoA substrates from simple carbon sources via de novo fatty acid synthesis, and thereby produce novel biodegradable polyesters having physical properties similar to those of petrochemical-derived plastics.
The present invention also provides a description of methods that would allow one skilled in the art to isolate, identify, and characterize genes that encode proteins involved in the process of converting lipid biosynthetic intermediates to PHA biosynthetic intermediates. In particular, methods are described for identifying genes that encode CoA-ACP acyltransferases that would be useful in the direct conversion of acyl-ACP to acyl-CoA for PHA biosynthesis.
Further scope of the applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the following detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.